Mutations in escherichia coli aceE and ribB genes allow survival of strains defective in the first step of the isoprenoid biosynthesis pathway

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway

Mutations in Escherichia coli aceE and ribB genes allow survival of strains defective in the first step of the isoprenoid biosynthesis pathway

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway

Hunting for Plant Nitric Oxide Synthase Provides New Evidence of a Central Role for Plastids in Nitric Oxide Metabolism

Nitric oxide (NO) has emerged as a central signaling molecule in plants and animals. However, the long search for a plant NO synthase (NOS) enzyme has only encountered false leads. The first works describing a pathogen-induced NOS-like plant protein were soon retracted. New hope came from the identification of NOS1, an Arabidopsis thaliana protein with an atypical NOS activity that was found to be targeted to mitochondria in roots. Although concerns about the NO-producing activity of this protein were raised (causing the renaming of the protein to NO-associated 1), compelling data on its biological role were missing until recently. Strong evidence is now available that this protein functions as a GTPase that is actually targeted to plastids, where it might be required for ribosome function. These and other results support the argument that the defective NO production in loss-of-function mutants is an indirect effect of interfering with normal plastid functions and that plastids play an important role in regulating NO levels in plant cells

A Mutant Impaired in the Production of Plastome-Encoded Proteins Uncovers a Mechanism for the Homeostasis of Isoprenoid Biosynthetic Enzymes in Arabidopsis Plastids[W]

The plastid-localized methylerythritol phosphate (MEP) pathway synthesizes the isoprenoid precursors for the production of essential photosynthesis-related compounds and hormones. We have identified an Arabidopsis thaliana mutant, rif1, in which posttranscriptional upregulation of MEP pathway enzyme levels is caused by the loss of function of At3g47450, a gene originally reported to encode a mitochondrial protein related to nitric oxide synthesis. However, we show that nitric oxide is not involved in the regulation of the MEP pathway and that the encoded protein is a plastid-targeted homolog of the Bacillus subtilis YqeH protein, a GTPase required for proper ribosome assembly. Consistently, in rif1 seedlings, decreased levels of plastome-encoded proteins were observed, with the exception of ClpP1, a catalytic subunit of the plastidial Clp protease complex. The unexpected accumulation of ClpP1 in plastids with reduced protein synthesis suggested a compensatory mechanism in response to decreased Clp activity levels. In agreement, a negative correlation was found between Clp protease activity and MEP pathway enzyme levels in different experiments, suggesting that Clp-mediated degradation of MEP pathway enzymes might be a mechanism used by individual plastids to finely adjust plastidial isoprenoid biosynthesis to their functional and physiological states

An enhancer trap system to track developmental dynamics in Marchantia polymorpha.

A combination of streamlined genetics, experimental tractability and relative morphological simplicity compared to vascular plants makes the liverwort Marchantia polymorpha an ideal model system for studying many aspects of plant biology. Here we describe a transformation vector combining a constitutive fluorescent membrane marker with a nuclear marker that is regulated by nearby enhancer elements and use this to produce a library of enhancer trap lines for Marchantia. Screening gemmae from these lines allowed the identification and characterization of novel marker lines, including markers for rhizoids and oil cells. The library allowed the identification of a margin tissue running around the thallus edge, highlighted during thallus development. The expression of this marker is correlated with auxin levels. We generated multiple markers for the meristematic apical notch region, which have different spatial expression patterns, reappear at different times during meristem regeneration following apical notch excision and have varying responses to auxin supplementation or inhibition. This reveals that there are proximodistal substructures within the apical notch that could not be observed otherwise. We employed our markers to study Marchantia sporeling development, observing meristem emergence as defining the protonema-to-prothallus stage transition, and subsequent production of margin tissue during the prothallus stage. Exogenous auxin treatment stalls meristem emergence at the protonema stage but does not inhibit cell division, resulting in callus-like sporelings with many rhizoids, whereas pharmacologically inhibiting auxin synthesis and transport does not prevent meristem emergence. This enhancer trap system presents a useful resource for the community and will contribute to future Marchantia research

Epidermal expression pattern of the auxin efflux carrier PIN1 in petals supports a divergent polarity field.

<p>(A) At 0 DAP, epidermal PIN1::GFP is preferentially localised to the distal end of cells in the plane of the petal. White arrows on cells point towards the centre of the distal PIN1 expression. (B, C) At 0.5 and 1.5 DAP, PIN1 polarity in the epidermis points divergently toward the distal end of the tissue. Strong expression is also seen at or near the petal margin but without clear polar localisation. (D) Deeper section of (C) showing expression in the provascular tissue. (E) At 3.5 DAP, PIN expression in the main plain of the epidermis is weak. (F) Enlargement of the white box in (E) showing PIN1 signal at the distal end of the epidermal cells. PIN1 expression is lower so the GFP channel is shown merged with the red channel (corresponding to the emission of chlorophyll autofluorescence) to facilitate visualisation. (G) At 4 DAP, PIN1 signal is no longer detected in the main plane of the petal but can be observed in the petal margin and vascular tissue. (H) Close up of (G). (I) At 6.5 DAP, expression at the distal margin has disappeared. (J) Little signal is observed from 7 DAP onwards. Width of petals: (A–C) 30, 45, and 75; (E) 146; (G) 170; (I) 343; (J) 425 µm. Scale bar, 20 µm (A–H), 50 µm (I), and 80 µm (J).</p

Broad distal distribution of auxin response marker <i>DR5::GFP</i> in the petal distal margin.

<p>(A–C) In leaf primordia, DR5 is expressed at the tip through early to later stages of development. DR5 is also expressed in the internal provasculature system (marked with a broken blue line in B). (D, E) In petal primordia at 0 and 0.5 DAP, DR5 is expressed at the distal tip. The orange line indicates the extent of the DR5 signal along the petal margin. (F, G) At 2–3 DAP, DR5 activity expands to a broader distal domain of the petal margin. (H) At 5 DAP the broad pattern of petal marginal expression is maintained. (I) At 7 DAP petal marginal expression narrows (the broken orange line marks the region where the DR5 domain first disappears). (J) By 10 DAP petal marginal expression has disappeared. DR5 signal in the internal provasculature system of the petal in (E–H). Width of leaves (A–C): 46, 80, and 150 µm. Width of petals (D–J): 26, 45, 82, 141, 261, 374, and 875 µm. Scale bar, 20 µm (A–G), 40 µm (H), 80 µm (I), and 250 µm (J).</p

Petal clonal analysis after induction at 0 DAP.

<p>(A) Sector map with shapes and positions of clones induced at 0 DAP and imaged 2 d after induction (at a width that corresponds to 2 DAP). Clones were imaged from dissected, flattened petals and warped to an average petal shape and overlaid (using a different colour for each petal). (B) Image of a distal epidermal clone (framed with a red box in A) and the corresponding outline of cells. (C) Sector map with clones induced at 0 DAP and imaged 4 d after induction (at a width that corresponds to 4 DAP). (D) Sectors imaged at 6 d (6 DAP). (E) Sectors imaged at 8 d (8 DAP). (F) Image of a distal epidermal clone (framed with a red box in E). (G) Enlargement of the yellow box in (F) showing some epidermal cells. (H) Sector map with clones induced at 0 DAP and imaged at 10 d after induction (10 DAP). (I) Sectors imaged at 12 d (12 DAP). The black arrow points to a distal clone. (J) Distal epidermal cells at 12 DAP at the position of the blue box in (I). (K) Proximal epidermal cells at the position of the violet box in (I). Images at (A, C–E, H, I) are scaled to the same length to allow the difference in clone shape to be compared. Scale bar, 5 µm (B, G), 10 µm (A, J), 20 µm (F, K), 50 µm (C), 100 µm (D, E), 200 µm (H), and 300 µm (I).</p

A less active and more discontinuous distal organiser in <i>jagged</i> mutants.

<p>(A) Initial canvas for divergent petal model (as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001550#pbio-1001550-g004" target="_blank">Figure 4D</a>) with no DGRAD activity (as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001550#pbio-1001550-g008" target="_blank">Figure 8A</a>, 0% model) and with a more discontinuous distal organiser activity as implemented by a discontinuous DISTORG (cyan) region. (B) Canvas at 2 and 12 DAP. Petals develop a jagged or serrated distal petal outline, with the tip of the serrations corresponding to regions with high DISTORG activity (as shown in the enlargement of the orange square at 12 DAP). (C) <i>DR5::GFP</i> signal is not detected at early stages in <i>jag-1</i> mutant, and (D, E) it is detected at later stages in several regions of the distal domain in a discontinuous way. (F) Enlargement of the orange square in (E). (G) <i>PIN1::PIN1:GFP</i> in <i>jag-1</i> mutant. PIN1 signal is reduced in the <i>jag-1</i> mutant at all stages and has a narrower distribution in the distal margin compared to wild-type petal (compare with <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001550#pbio-1001550-g006" target="_blank">Figure 6D</a>). (H) <i>PIN3::PIN3:GFP</i> in <i>jag-1</i> mutant shows reduced expression, and it is more discontinuous in the distal margin (compare with wild type in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001550#pbio-1001550-g007" target="_blank">Figure 7D</a>). Width of petals: (C–G) 123, 310, 515, 66, and 84 µm. Scale bar, 10 µm (A, B) (2 DAP), 20 µm (C, G, H), 100 µm (D), 200 µm (E).</p